Nanoscale electrostatic gating of molecular transport through nuclear pore complexes as probed by scanning electrochemical microscopy

The nuclear pore complex (NPC) uses positive residues of amino acids to electrostatically regulate molecular transport through the peripheral route.

Micropore-Supported NE. The nucleoplasm-free NE of nucleus isolated from a Xenopus laevis oocyte was supported by a microporous Si 3 N 4 membrane as reported recently. S4 The permeability of micropore-supported NE patches was measured by polyion-selective micropipet tips S5 without fouling, which was caused by small proteins leached from the nucleoplasm. S6,S7 A large nucleus (~0.4 mm in diameter) was isolated from the stage VI oocyte of an adult female Xenopus laevis frog (NASCO, Fort S3 Atkinson, WI, USA) in the isotonic 1.5% PVP solution of mock intracellular buffer (MIB) at pH 7.4 containing 90 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , 1.1 mM EGTA, 0.15 mM CaCl 2 , and 10 mM HEPES, where free Ca 2+ was buffered at the physiological level of ~200 nM in oocytes. S8 The nucleus was swollen in a hypotonic MIB solution of 0.55% PVP to detach the NE from the nucleoplasm. The NE was spread over by using an insect pin (26002-10, Fine Science Tools, Foster City, CA, USA) bent to a 0.5-0.6 mm length using tweezers under a stereomicroscope and attached to a 5 cm-long borosilicate glass capillary (B100-58-10, Sutter Instruments, Novato, CA) using UV-curable glue (OP-4-20632, Dymax, Torrington, CT, USA). The spread NE was adhered to a microporous membrane treated with Cell-Tak as an adhesive. The hypotonic MIB solution was replaced with transport media for SECM studies, i.e., a PVP-free MIB solution or a low salt buffer (LSB) containing 1 mM KCl, 0.5 mM MgCl 2 , and 10 mM HEPES at pH 7.5. S9

SECM.
A home-built SECM instrument S10 was combined with a potentiostat (CHI 900A, CH Instruments, Austin, TX, USA) and was controlled by using a custom Labview program (National Instruments, Austin, TX). A micropore-supported NE was set up in the SECM cell as shown in Fig. S1.
The tapered end of a micropipet was milled by the focused ion beam (FIB) of gallium ions (30 keV) at 100 pA using a dual beam instrument (Scios, FEI, Hillisboro, OR, USA) until a tip inner diameter of 1 µm was obtained for small monovalent ions and characterized by scanning electron microscopy. S5,S7 An 3 µm-diameter tip was obtained for polyions and small monovalent ions by using 5 nA beam for bulk milling and 100 pA beam for smoothening and was characterized also by scanning electron microscopy.
FIB-milled glass micropipets were reacted with chlorotrimethylsilane in a vacuum-dried desiccator 11 and filled with a NB solution of organic electrolytes. A potential was applied to an Ag wire in the S4 organic electrolyte solution against an aqueous Ag/AgCl reference electrode to drive ion transfer across the micropipet-supported liquid/liquid interface by using a Pt wire as an aqueous counter electrode.
SECM imaging employed a lateral tip scan rate of 1 µm/s and a tip step size of 0.5 µm for both directions. An SECM approach curve was measured by vertically bringing the tip to the center of NE patch at a scan rate of 0.50 µm/s. These scan rates were slow enough to obtain steady-state tip currents.

S6
Finite Element Simulation. The limiting current at a disk-shaped tip in the SECM configuration was simulated by solving an axisymmetric (2D) diffusion problem as defined in a cylindrical coordinate (Fig. S3). The origin of the axes was set at the center of the upper orifice of a micropore. Initially, the solution phase contains a redox probe at a bulk concentration of c 0 . The steady-state diffusion of a redox probe in solution was defined by (S1) where c N and c C are concentrations of the redox probe at (r, z) in solutions at nucleoplasmic and cytoplasmic sides of the NE, respectively, and D w is the diffusion coefficient of the probe. The probe was electrolyzed at the tip to yield a diffusion-limited current, i T , based on the boundary condition given by c = 0 (S3) A boundary condition for the redox probe at the nuclear envelope was given by eq 2 to yield Other boundary conditions for the redox probe are given in Fig. S3. We assumed that the diffusion coefficient of the product of the tip reaction was identical to that of the probe, D w . Accordingly, the diffusion problem was defined and solved only for the redox probe.
In addition, geometric parameters were defined by using dimensionless parameters as (S12) L  d a This problem was solved numerically to calculate the normalized tip current, i T /i T,∞ , which was set to 1 at L = 25.

Approach Curves of Small Monovalent Ions.
We measured approach curves of small monovalent ions at the micropore-supported NEs by using micropipet-supported tips to determine the corresponding NE permeability. Either 1 or 3 µm-diameter micropipet tips were filled with the DCE solution of 0.1 M TDDATFAB to construct the following electrochemical cell Ag|AgCl|0.3 mM monovalent ions in LSB or MIB|0.1 M TDATFAB in DCE|Ag

S9
Approach curves of probe ions (e.g., TBA + in Fig. S4) were measured at the NE patches supported with 3 and 10 µm-diameter pores of Si 3 N 4 membranes in LSB or MIB by using 1 or 3 µm-diameter tips, respectively.